Quinazolinone containing pharmaceutical compositions for prevention of neovascularization and for treating malignancies

ABSTRACT

The invention provides a composition for attenuating neovascularization and treating malignancies, including a pharmaceutically effective amount of a compound having a formula: ##STR1## wherein: R 1  is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy; 
     R 2  is a member of the group consisting of hydroxy, acetoxy, and lower alkoxy, and 
     R 3  is a member of the group consisting of hydrogen and lower alkenoxy carbonyl; 
     as active ingredient therein, in combination with a pharmaceutically acceptable carrier.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to compositions containing quinazolinones.More particularly, the present invention relates to a composition,containing a quinazolinone derivative, useful for the treatment ofangiogenic-associated diseases, as well as for the treatment ofmalignancies, including inhibition of primary tumor growth, tumorprogression and metastasis.

Malignancies arc characterized by the growth and spread of tumors. Anumber of factors are important in the progression of this disease. Onecrucial factor is angiogenesis, a complex process in which capillaryblood vessels grow in an ordered sequence of events [J. Folkman and M.Klagsbrun, Science, Vol. 235, pp 442-447 (1987); J. Folkman and Y.Shing, J. Biol. Chem., Vol. 267, pp. 10931-10934 (1992)]. Once a tumorhas started, every increase in tumor cell population must be preceded byan increase in new capillaries that converge on the tumor and supply thecells with oxygen and nutrients [J. Folkman, Perspect. in Biol. andMed., Vol 29, p. 10-36 (1985); J. Folkman, J. Natl. Cancer Inst., Vol.82, pp. 4-6 (1989); N. Weidner, et al., Amer. J. Pathol., Vol. 143, pp.401-409 (1993)]. Tumors may thus remain harmless and confined to theirtissue of origin, as long as an accompanying angiogenic program isprevented from being activated. Since the angiogenesis-dependent step intumor progression is shared by solid tumors of all etiologies, theability to inhibit tumor-associated angiogenesis is a most promisingapproach in combating cancer [M. S. O'Reilly, et al., Cell, Vol. 79, pp.316-328 (1994)].

A substantial body of experimental evidence supports the hypothesis thattumor angiogenesis is fundamental for the growth and metastasis of solidtumors [J. Folkman, ibid. (1989); N. Weidner, et al., ibid. (1993); M.S. O'Reilly, et al., ibid. (1994); N. Weidner, et al., N. Eng. J. Med.,Vol. 324, pp. 1-8 (1991)]. Indeed, the majority of solid tumors are noteven clinically detectable until after the occurrence ofneovascularization, whose induction in solid tumors is mediated by oneor more angiogenic factors [J. Folkman, ibid. (1987); J. Folkman and Y.Shing, ibid. (1992)].

Furthermore, angiogenesis is also important in a number of otherpathological processes, including arthritis, psoriasis, diabeticretinopathy, chronic inflammation, scleroderma, hemangioma, retrolentalfibroplasia and abnormal capillary proliferation in hemophiliac joints,prolonged menstruation and bleeding, and other disorders of the femalereproductive system [J. Folkman, Nature Medicine, Vol 1, p. 27-31,(1995); J. W. Miller, et al., J. Pathol., Vol. 145, pp. 574-584 (1994);A. P. Adamid, et al., Amer. J. Ophthal., Vol. 118, pp. 445-450 (1994);K. Takahashi, at al., J. Clin. Invest., Vol.93, pp.2357-2364 (1994); D.J. Peacock, et al., J. Exp. Med., Vol. 175, pp. 1135-1138 (1992); B. J.Nickoloff, et al., Amer. J. Pathol., Vol. 44, pp. 820-828 (1994); J.Folkman, Steroid Hormones and Uterine Bleeding, N. J. Alexander and C.d'Arcangues, Eds., American Association for the Advancement of SciencePress, Washington, D.C., U.S.A., pp. 144-158 (1992)].

Thus, clearly methods of blocking the mechanism of angiogenesis arenecessary. The basic mechanism of angiogenesis is as follows. Briefly,when a new capillary sprout grows from the side of the venule,endothelial cells degrade basement membrane, migrate toward anangiogenic source, proliferate, form a lumen, join the tips of twosprouts to generate a capillary loop, and manufacture new basementmembrane [J. Folkman, Perspectives in Biology and Medicine, Vol. 29, pp.1-36 (1985)].

Degradation and remodeling of the ECM are essential processes for themechanism of angiogenesis. In addition, ECM components synthesized byendothelial cells (i.e., collagens, laminin, thrombospondin, fibronectinand SPARC) function to regulate endothelial cell growth, migration andshape [J. Bischoff, Trends Cell Biol., No. 5, pp. 69-74 (1995)]. Bovineaortic endothelial cells (BAE) undergoing sprouting and tube formationsynthesize type I collagen and SPARC. It was proposed that type Icollagen may be involved in directing migration and assembly of the BAEcells [M. L. Iruela-Arispe, et al., Lab. Invest., No. 64, pp. 174-186(1991). It was also found that exogenous type I collagen promoted rapidtube formation by confluent human dermal microvascular endothelial cells[C. J. Jackson and K. L. Jenkins, Exp. Cell Res., No. 192, pp. 319-323(1991)]. The tubes contained collagen fibrils in the luminal spaces,suggesting that the endothelial cells use the fibrils to fold and aligninto tube structures.

Furthermore, in order to extend a capillary blood vessel, interactionsmust occur between ECM components and the surrounding matrix molecules,which provide a scaffold for the ECM components of the new vessel[Brooks, P. C. et al., Cell, Vol 79, p. 1157-1164, (1994)]. Disruptionof cell-matrix interactions induced apoptosis in human endothelialcells. It has been demonstrated that integrin α₂ β₃, which has anenhanced expression in angiogenic vascular cells, promotes a survivalsignal, since inhibitors of this integrin cause unscheduled apoptosisand disintegration of newly formed blood vessels.

In order to treat angiogenesis-related diseases, several inhibitors ofthe above mechanism of angiogenesis are being studied, includingplatelet factor 4, the fumagillin derivative AGM 1470, Interferon α₂ a,thrombospondin, angiostatic steroids, and angiostatin [J. Folkman,ibid., (1995); M. S. O'Reilly, et al., ibid. (1994); V. Castle, et al.,J. Clin. Invest., Vol. 87, pp.1183-1888; D. Ingber, et al., Nature, Vol.348, pp. 555-557]. All of these compounds have disadvantages. Forexample, endostatin and angiostatin are proteins, so that they have allof the disadvantages of proteins, including the requirement for beingadministered parenterally. Therefore, a non-protein inhibitor whichwould selectively block the underlying mechanism of angiogenesis withoutadversely affecting other physiological functions, and which could beadministered by many different routes, would be extremely useful.

In addition, many of the compounds that are now being evaluated asantiangiogenic agents are proteins, e.g., antibodies, thrombospondin,angiostatin, platelet factor IV [J. Folkman, ibid. (1995); M. S.O'Reilly, et al., ibid. (1994); V. Castle, et al., ibid., P. C. Brooks,et al., ibid. (1994)], which suffer from poor bioavailability and arereadily degraded in the body. Hence, these substances should beadministered in high doses and frequencies.

Other approaches for cancer treatment focus on cytotoxic therapies, suchas chemotherapy or radiation treatments, in order to kill activelyproliferating cells. Unfortunately, these therapies are highly toxic tonon-cancer cells and cause severe side effects, such as bone marrowsuppression, hair loss and gastrointestinal disturbances.

As noted above, degradation and remodeling of the ECM are essentialprocesses for the mechanism of angiogenesis. Such processes involve thesynthesis of a number of components of the ECM, such as collagen. Thesynthesis of collagen is also involved in a number of other pathologicalconditions. For example, clinical conditions and disorders associatedwith primary or secondary fibrosis, such as systemic sclerosis,graft-versus-host disease (GVHD), pulmonary and hepatic fibrosis and alarge variety of autoimmune disorders, are distinguished by excessiveproduction of connective tissue, which results in the destruction ofnormal tissue architecture and function. These diseases can best beinterpreted in terms of perturbations in cellular functions, a majormanifestation of which is excessive collagen synthesis and deposition.The crucial role of collagen in fibrosis has prompted attempts todevelop drugs that inhibit its accumulation [K. J. Kivirikko, Annals ofMedicine, Vol.25, pp. 113-126 (1993)].

Such drugs can act by modulating the synthesis of the procollagenpolypeptide chains, or by inhibiting specific post-translational events,which will lead either to reduced formation of extra-cellular collagenfibers or to an accumulation of fibers with altered properties.Unfortunately, only a few inhibitors of collagen synthesis areavailable, despite the importance of this protein in sustaining tissueintegrity and its involvement in various disorders.

For example, cytotoxic drugs have been used in an attempt to slow theproliferation of collagen-producing fibroblasts [J. A. Casas, et al.,Ann. Rhem. Dis., Vol. 46. p. 763 (1987)], such as colchicine, whichslows collagen secretion into the extracellular matrix [D. Kershenobich,et al., N. Engl. J. Med., Vol. 318, p. 1709 (1988)], as well asinhibitors of key collagen metabolism enzymes (K. Karvonen, et al., J.Biol. Chem., Vol. 265, p. 8414 (1990); C. J. Cunliffe, et al., J. Med.Chem., Vol. 35, p.2652 (1992)].

Unfortunately, none of these inhibitors are collagen-type specific.Also, there are serious concerns about the toxic consequences ofinterfering with biosynthesis of other vital collagenous molecules, suchas Clq in the classical complement pathway, acetylcholine esterase ofthe neuro-muscular junction endplate, conglutinin and pulmonarysurfactant apoprotein.

Other drugs which can inhibit collagen synthesis, such as nifedipine andphenytoin, inhibit synthesis of other proteins as well, therebynon-specifically blocking the collagen biosynthetic pathway [T. Salo, etal., J. Oral Pathol. Med., Vol. 19, p. 404 (1990)].

Collagen cross-linking inhibitors, such as β-amino-propionitrile, arealso non-specific, although they can serve as useful anti-fibroticagents. Their prolonged use causes lathritic syndrome and interfereswith elastogenesis, since elastin, another fibrous connective tissueprotein, is also cross-linked. In addition, the collagen cross-linkinginhibitory effect is secondary, and collagen overproduction has toprecede its degradation by collagenase. Thus, a type-specific inhibitorof the synthesis of collagen itself is clearly required as ananti-fibrotic agent.

Such a type-specific collagen synthesis inhibitor is disclosed in U.S.patent application Ser. No. 08/181,066 for the treatment of a fibroticcondition, restenosis or glomerulosclerosis. This specific inhibitor isa composition with a pharmaceutically effective amount of apharmaceutically active compound of a formula: ##STR2## wherein: R₁ is amember of the group consisting of hydrogen, halogen, nitro, benzo, loweralkyl, phenyl and lower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl. Of this group of compounds, Halofuginone has beenfound to be particularly effective for such treatment.

U.S. Pat. No. 5,449,678 discloses that these compounds are effective inthe treatment of fibrotic conditions such as scleroderma and GVHD. WONo. 96/06616 further discloses that these compounds are effective intreating restenosis. The two former conditions are associated withexcessive collagen deposition, which can be inhibited by Halofuginone.Restenosis is characterized by smooth muscle cell proliferation andextracellular matrix accumulation within the lumen of affected bloodvessels in response to a vascular injury [Choi et al, Arch. Surg., Vol.130, p. 257-261 (1995)]. One hallmark of such smooth muscle cellproliferation is a phenotypic alteration, from the normal contractilephenotype to a synthetic one. Type I collagen has been shown to supportsuch a phenotypic alteration, which can be blocked by Halofuginone [Choiet al., Arch. Surg., Vol. 130, p. 257-261 (1995); U.S. Pat. No.5,449,678]. Thus, Halofuginone can prevent such differentiation ofsmooth muscle cells after vascular injury by blocking the synthesis oftype I collagen. Other in vitro studies show that Halofuginone can alsoinhibit the proliferation of 3T3 fibroblast cells [U.S. Pat. No.5,449,678].

However, the in vitro action of Halofuginone does not always predict itsin vivo effects. For example, Halofuginone inhibits the synthesis ofcollagen type I in bone chrondrocytes in vitro, as demonstrated in U.S.Pat. No. 5,449,678. However, chickens treated with Halofuginone were notreported to have an increased rate of bone breakage, indicating that theeffect is not seen in vivo. Thus, the exact behavior of Halofuginone invivo cannot always be predicted from in vitro studies.

Furthermore, the ability of Halofuginone or other related quinolinonesto block or inhibit physiological processes related to tumor growth andprogression is not known in the prior art. Although Halofuginone hasbeen shown to have a specific inhibitory effect on the synthesis of typeI collagen, such inhibition has not been previously shown to slow orhalt tumor progression, particularly in vivo.

There is thus a widely recognized unmet medical need for an inhibitor oftumor progression which is particularly effective in vivo, substantiallywithout adversely affecting other physiological processes.

SUMMARY OF THE INVENTION

Unexpectedly, it has been found, as described in the examples below,that Halofuginone can also slow or halt tumor progression in vivo,possibly by inhibiting angiogenesis, or by blocking ECM deposition, orpossibly through both mechanisms, although another mechanism ormechanisms could also be responsible. While inhibition of angiogenesisand of ECM deposition, or a combination thereof, are proposed asplausible mechanisms, it is not desired to be limited to a singlemechanism, nor is it necessary since the in vivo data presented belowclearly demonstrate the efficacy of Halofuginone as an inhibitor oftumor progression in vivo.

According to an embodiment of the present invention, there is provided acomposition for treating a tumor, including a pharmaceutically effectiveamount of a compound in combination win a pharmaceutically acceptablecarrier, the compound being a member of a group having a formula:##STR3## wherein: R₁ is a member of the group consisting of hydrogen,halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy, and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and lower alkenoxy.

According to another embodiment of the present invention, there isprovided a method of manufacturing a medicament for treating a tumor,including the step of placing a pharmaceutically effective amount of acompound in a pharmaceutically acceptable carrier, the compound being amember of a group having a formula: ##STR4## wherein: R₁ is a member ofthe group consisting of hydrogen, halogen, nitro, benzo, lower alkyl,phenyl, and lower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy, and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and louveralkenoxy-carbonyl.

According to yet another embodiment of the present invention, there isprovided a method of manufacturing a medicament for substantiallyinhibiting neovascularization, including the step of placing apharmaceutically effective amount of a compound in a pharmaceuticallyacceptable carrier, the compound being a member of a group having aformula: ##STR5## wherein: R₁ is a member of the group consisting ofhydrogen, halogen, nitro, benzo, lower alkyl, phenyl, and lower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy, and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl.

According to still another embodiment of the present invention, there isprovided a method for the treatment of angiogenesis in a subject,including the step of administering a pharmaceutically effective amountof a compound having a formula: ##STR6## wherein: R₁ is a member of thegroup consisting of hydrogen, halogen, nitro, benzo, lower alkyl,phenyl, and lower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl.

According to still another embodiment of the present invention there isprovided a method for the treatment of a tumor in a subject, includingthe step of administering a pharmaceutically effective amount of acompound having a formula: ##STR7## wherein: R₁ is a member of the groupconsisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl andlower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl.

There is also provided a composition for inhibiting cell proliferationenabled by a deposition of an extracellular matrix, including apharmaceutically effective amount of a compound having a formula:##STR8## wherein: R₁ is a member of the group consisting of hydrogen,halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;

R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy, and

R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl.

Preferably, the specific compound in each of the above embodiments isHalofuginone.

While the invention will now be described in connection with certainpreferred embodiments in the following figures and examples so thataspects thereof may be more fully understood and appreciated, it is notintended to limit the invention to these particular embodiments. On thecontrary, it is intended to cover all alternatives, modifications andequivalents as may be included within the scope of the invention asdefined by the appended claims. Thus, the following figures and exampleswhich include preferred embodiments will serve to illustrate thepractice of this invention, it being understood that the particularsshown are by way of example and for purposes of illustrative discussionof preferred embodiments of the present invention only, and arepresented in the cause of providing what is believed to be the mostuseful and readily understood description of formulation procedures aswell as of the principles and conceptual aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

FIGS. 1A-1F illustrate the inhibition and apoptosis of in vivo tumorgrowth in mice by Halofuginone;

FIGS. 2A and 2B show the effect of Halofuginone on ³ H-thymidineincorporation into bovine aortic endothelial cells maintained inculture, in the absence or the presence of bovine fibroblast growthfactor (bFGF);

FIGS. 3A and 3B illustrate the inhibitory effect of Halofuginone on theorganization of bovine aortic endothelial cells into capillary-likenetworks;

FIGS. 4A and 4B pictorially illustrate the inhibitory effect ofHalofuginone on microvessel formation from rat aortic rings embedded intype I collagen gel;

FIG. 5 is a dose-response curve of the inhibitory effect of Halofuginoneon microvessel formation, using the collagen Type I embedded rat aorticrings of FIGS. 4A and 4B;

FIG. 6 demonstrates the reversibility of the inhibitory effect ofHalofuginone;

FIG. 7 shows the effect of Halofuginone on sulfate incorporation intothe subendothelial ECM with bovine corneal endothelial cells;

FIGS. 8A-8D compare the effect of Halofuginone on incorporation ofsulfate, proline, lysine and glycine into the ECM by bovine cornealendothelial cells;

FIGS. 9A-9D illustrate the effect of Halofuginone on sulfate and glycineincorporation into the ECM of rat mesengial cells;

FIGS. 10A and 10B illustrate the inhibitory effect of Halofuginone on invivo neovascularization in the eyes of mice;

FIGS. 11A and 11B illustrate the effect of Halofuginone on ³ H-thymidineincorporation and proliferation of human leiomyosarcoma tumor cells; and

FIG. 12 illustrates the inhibition of collagen I gene expression byHalofuginone.

DESCRIPTION OF PREFERRED EMBODIMENTS

Unexpectedly, it has been found, as described in the examples below,that Halofuginone can also slow or halt tumor progression in vivo,possibly by inhibiting angiogenesis or by substantially completelyinhibiting deposition of ECM components, or a combination thereof,although another mechanism or mechanisms could also be responsible.Indeed, irrespective of the specific mechanism, the data presented belowclearly demonstrate the efficacy of Halofuginone in vivo at inhibitingtumor progression.

Such a finding is unexpected for three reasons. First, the behavior ofHalofuginone in vitro does not exactly correspond to its behavior invivo. This can be demonstrated by the differential effect ofHalofuginone observed with bone chondrocytes in vivo and in vitro.Halofuginone inhibits the synthesis of collagen type I in chrondrocytesin vitro, as demonstrated in U.S. Pat. No. 5,449,678. However, chickenstreated with Halofuginone were not reported to have an increased rate ofbone breakage, indicating that the effect is not seen in vivo. Thus, theexact behavior of Halofuginone in vivo cannot always be predicted fromin vitro studies.

Second, the only previously known examples of the inhibition of cellproliferation by Halofuginone involved either smooth muscle cells whichhad become phenotypically altered in response to a vascular injury, or3T3 fibroblasts [WO No. 96/06616 and Choi et al., Arch. Surg., Vol. 130,p. 257-261 (1995)]. These cells simply proliferated withoutorganization. By contrast, angiogenesis involves the formation of highlyorganized vascular structures. Thus, the finding that Halofuginone caninhibit such angiogenesis is both novel and non-obvious.

Furthermore, the examples given below clearly demonstrate thatHalofuginone is also effective in the inhibition of cell proliferationenabled by the deposition of an extracellular matrix, in vivo as well asin vitro. Such specific inhibition has never been demonstrated before,particularly in vivo.

Thus, nothing in the prior art taught or suggested that Halofuginonewould be useful in the treatment of malignancies in vivo. Furthermore,the ability of Halofuginone, and related compounds, to slow or halttumor progression, and to inhibit cell proliferation enabled by thedeposition of an extracellular matrix, is both novel and non-obvious.The demonstration of such an ability in vivo is particularly unexpected,given the differential responses seen in vitro and in vivo toHalofuginone.

The present invention may be more readily understood with reference tothe following illustrative examples and figures. It should be noted thatalthough reference is made exclusively to Halofuginone, it is believedthat the other quinazolinone derivatives described and claimed in U.S.Pat. No. 3,320,124, the teachings of which are incorporated herein byreference, have similar properties.

EXAMPLE 1 Inhibition of In Vivo Tumor Growth in Mice by Halofuginone

The inhibition of growth of two different tumors was examined in vivo inmice. The first type of tumor was T50 bladder carcinoma in C3H mice, andthe second type was EHS sarcoma in C57BL/6 mice. Unexpectedly,Halofuginone was shown to have a significant inhibitory effect on tumorgrowth and progression in these in vivo models. Quantitative results areshown in FIGS. 1A, 1B and 1C. A photograph of control and halofuginonetreated mice is presented in FIG. 1D. Photographs of the tumors excisedfrom control mice and mice fed with halofuginone containing diet (5 and10 mg/kg food) are presented in FIG. 1E. Histological sections of thetumors are presented in FIG. 1F.

C3H mice were divided into two groups (6 mice each). The experimentalgroup received a diet containing 5 mg/kg of Halofuginone 3 days prior tothe injection of T50 bladder carcinoma cells and during 2 weeks after.Cultured T50 cells, a more aggressive variant of the chemically inducedMBT2 mouse bladder carcinoma, were dissociated with trypsin/EDTA into asingle cell suspension (10⁶ cells/ml) in growth medium and inoculateds.c. in two sites on the dorsa of mice. The right side received 0.4*10⁵cells, and the left side received 2*10⁵ cells. The tumor size wasestimated by measurement of tumor length in two directions, using theformula V=LW² /2, where V is volume, L is length and W is width. At theend of the experiment at day 17, the mice were weighed, the tumors wereexcised and a sample of the tumor tissue was fixed and processed forhistological examination

Tumor size in C3H mice which were fed with Halofuginone wassignificantly reduced, by about 70-80%, as compared to control micemaintained on a normal diet, as shown in FIGS. 1A and 1B. The anti-tumoreffect of Halofuginone was observed both at the high tumor cell dose,with a volume of 5.0±3.07 cm³ for control mice and 1.0±0.92 cm³ forHalofuginone containing diet, and the low tumor cell dose, with a volumeof 1.63±0.98 cm³ for control mice and 0.29±0.28 cm³ for Halofuginone fedmice. Furthermore, the overall weight of the Halofuginone treated C3Hmice was lower than the weight of the untreated mice, 24±3.1 g and40±3.8 g, respectively, due to the lower tumor burden. Data for all miceare shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Effect of Halofuginone on T50 Tumor Size (cm.sup.3)                             Sample  - Halofuginone    + Halofuginone                                    No.   0.4 * 10.sup.5 cells                                                                     2 * 10.sup.5 cells                                                                       0.4 * 10.sup.5 cells                                                                   2 * 10.sup.5 cells                       ______________________________________                                        1     2.6        4.3        0.036    0.7                                        2 2.7 10.1 0.064 0.1                                                          3 0.4 6.1 0.150 1.1                                                           4 1.0 6.6 0.730 2.0                                                           5 0.9 0.7 0.510 1.1                                                           6 2.2 2.2 NA NA                                                             ______________________________________                                    

Animals bearing Engelbreth-Holm-Swarm (EHS) tumors were sacrificed andthe tumor tissue was excised and minced under aseptic conditions. TheEHS tumor is characterized by a large amount of basement membranecomponents. A suspension of the tumor tissue in 0.2 ml of PBS wasinjected subcutaneously in the dorsal posterior region of C57BL/6 malemice. The mice were divided into two groups of 10 mice each. The animalsof the experimental group were fed with a diet containing 5 mg/kgHalofuginone for 3 days prior to tumor injection and during 3 weeksafter. The tumor size was estimated by measurement of tumor length intwo directions, using the formula V=LW² /2. On the final day of theexperiment, i.e., 20 days after tumor injection, the mice were weighed,the tumors were excised and a sample of the tumor tissue was fixed andprocessed for histological examination.

Tumor size in C57BL/6 mice which were fed Halofuginone was reduced byabout 75% as compared to control mice, with volumes of 1.72±1.85 cm³ and8.15±4.88 cm³, respectively, as shown in FIG. 1C. The anti-tumor effectof Halofuginone was reflected by the nearly normal weight ofHalofuginone-fed mice, 19.7±0.47 g, as opposed to approximately 1.6 foldhigher weight of control mice, 33.3±2.49 g, due to the tumor burden.Data for all mice are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Effect of Halofuginone on EHS Tumor Size                                        Sample No.     - Halofuginone                                                                           + Halofuginone                                    ______________________________________                                        1            12.6       1.3                                                     2 5.8 1.2                                                                     3 3.2 1.3                                                                     4 12.6 1.7                                                                    5 4.9 0                                                                       6 6.8 0                                                                       7 4.9 2.2                                                                     8 18.9 0.04                                                                   9 2.8 6.5                                                                     10 9.0 3.0                                                                  ______________________________________                                    

Histological sections of the tumors were subjected to tunnel staining[Gavrieli, Y. et al. J. Cell Biol. Vol. 119. pp 493-501, 1992], in orderto evaluate the extent of tumor cell necrosis and apoptosis. Asdemonstrated in FIG. 1F panel B, the majority of cells in tumor sectionsderived from halofuginone treated mice exhibited an intense staining, ascompared to only a small percentage of the cells that were stained insections derived from tumors in untreated mice (FIG. 1F, panel A). Thehigh degree of cell apoptosis and necrosis in halofuginone treatedtumors is in all likelihood due to an inhibition of angiogenesis and ECMdeposition, both critical factors supporting growth and survival of thetumor cells.

EXAMPLE 2 Halofuginone-Induced Inhibition of ³ H-Thymidine Incorporationinto Vascular Endothelial Cells

Cultures of vascular endothelial cells were established from bovineaorta as previously described [D, Gospodarowicz, et al., Proc. Natl.Acad. Sci. U.S.A., Vol. 73, p. 4120 (1979)]. Bovine aortic endothelialcells were plated (4×10⁴ cells/16 mm well) in DMEM (1 g glucose/liter)supplemented with 10% calf serum, 50 U/ml penicillin, and 50 μg/mlstreptomycin at 37° C. in 10% CO₂ humidified incubators.

Four days after plating, the subconfluent cells were exposed toincreasing concentrations of Halofuginone (100-500 ng/ml), in theabsence or presence of 1 ng/ml bFGF. ³ H-thymidine (1 μCi/well) was thenadded for an additional 48 hours, and DNA synthesis was assayed bymeasuring the radioactivity incorporated into trichloroacetic acidinsoluble material [M. Benezra, et al., Cancer Res., Vol. 52, pp.5656-5662 (1992); I. Vlodavsky, et al., Proc. Natl. Acad. Sci. U.S.A.,Vol. 84, pp. 2292-2296 (1987)].

FIG. 2A shows the results obtained in the absence of bFGF, while FIG. 2Bshows the results obtained in the presence of bFGF. As demonstrated inFIGS. 2A and 2B, 50% inhibition of ³ H-thymidine incorporation wasobtained at 100 ng/ml Halofuginone, regardless of whether or not bFGF (1ng/ml) was added to the culture medium.

EXAMPLE 3 Organization of Endothelial Cells into Capillary-Like Networks

Halofuginone was found to prevent the organization of endothelial cellsinto a defined structure, and specifically inhibited the organization ofthese cells into capillary-like networks. Results are shown in FIGS. 3Aand 3B.

Type I collagen was prepared from the tail tendons of adultSprague-Dawley rats. Briefly, the collagen fibers were solubilized by aslow siring for 48 h at 4° C. in a sterile, 1/1000 (v/v) acetic acidsolution (300 ml for 1 g of collagen). The resulting solution wasfiltered trough a sterile triple gauze and centrifuged at 16,000 g for 1h at 4° C. The supernatant was then extensively dialyzed against 1/10DMEM and stored at 4° C. The collagen matrix gel was obtained bysimultaneously raising the pH and ionic strength of the collagensolution. For this purpose, 7 vol of collagen solution were quicklymixed with 1 vol of 10× Minimum Essential Medium and 2 vol sodiumbicarbonate (0.15M) [R. F. Nicosa and A. Ottinetti, Lab. Invest., Vol.63, pp. 115-122 (1990)].

Bovine aortic endothelial cells were seeded into 16 mm wells of a24-well plate and allowed to grow for 24 h, to obtain a subconfluentmonolayer. The culture medium was then removed and 0.4 ml of the coldcollagen mixture described above were poured on top of the cellmonolayer and allowed to polymerize for 10 min at 37° C. Fresh medium(0.6 ml), containing bFGF (1 ng/ml) and heparin (1 μg/ml), with (FIG.2B) or without (FIG. 2A) 0.1 μg/ml of Halofuginone, was added after thecollagen had gelled. The reorganization of the endothelial cellmonolayer was monitored and photographed with a Zeiss inverted phasecontrast photomicroscope [R. Monesano, et al., J. Cell Biol., Vol. 97,pp. 1648-1652 (1983)].

FIG. 3A illustrates the organization of endothelial cells intocapillary-like networks. Such organization is inhibited by Halofuginone,as demonstrated by FIG. 3B. Halofuginone completely inhibited theinvasion of the endothelial cells into the collagen gel and theirsubsequent organization into a network of branching and anastomosingcapillary-like tubes.

EXAMPLE 4 Microvessel Formation

Halofuginone was shown to inhibit microvessel formation from rings ofaortic tissues taken from rats. This effect was also shown to bereversible upon removal of Halofuginone. Results are shown in FIGS. 4A,4B, 5 and 6.

Thoracic aortas were obtained from 1- to 2-month-old SD (Sprague-Dawley)rats sacrificed by decapitation [R. F. Nicosia and A. Ottinetti, Lab.Invest., Vol. 63, pp. 115-122 (1990)]. The aortas were immediatelytransferred to a Petri dish with PBS. The fibro-adipose tissue aroundthe aorta was carefully removed under a dissecting microscope, and 1mm-long aortic rings were sectioned and extensively rinsed in PBS.

Type I collagen solution (0.2 ml) was added to each 16-mm well andgellation was allowed for 15 min at 37° C. Each aorta ring wastransferred and positioned to the center of the gel and another 0.4 mlof the collagen solution was carefully poured on top of the ring. Afterthe gel was formed, 0.4 ml of serum-free, endothelial growth medium,with or without 0.1 μg/ml Halofuginone, was added and the medium waschanged every other day.

FIG. 4A shows the culture at day 10, when the newly-formed branchingmicrovessels were developed from the end of resection of the aorta,giving rise to loops and networks. FIG. 4B shows the same culturetreated with 0.1 μg/ml halofuginone, replaced every other day. Underthese conditions, single cells were migrating from the aortic ringtoward the periphery, but failed to align into microvessel tubes.

FIG. 5 shows this effect quantitatively, at increasing doses ofHalofuginone. An almost complete inhibition of microvessel formation wasobtained at 100 ng/ml Halofuginone. Complete inhibition was observed inthe presence of 250 ng/ml Halofuginone. This effect was reversed uponremoval of the drug on day 2, as shown in FIG. 6. Such removal resultedin microvessel formation, similar to that seen with untreated aorticrings.

EXAMPLE 5 Halofuginone Inhibition of Sulfate Incorporation into ECM ofCultured Endothelial Cells

Halofuginone was shown to have an inhibitory effect on the deposition ofECM (extracellular matrix components), as shown in FIG. 7 and in otherexamples below.

Cultures of bovine corneal endothelial cells were established from steereyes and maintained as previously described [D. Gospodarowicz, et al.,Exp. Eye Res., No. 25, pp. 75-89 (1977)]. Cells were cultured at 37° C.in 10% CO₂ humidified incubators and the experiments were performed withearly (3-8) cell passages.

For preparation of sulfate-labelled ECM (extra-cellular matrix), cornealendothelial cells were seeded into 4-well plates at a confluent densityforming, within 4-6 h, a contact inhibited cell monolayer composed ofclosely apposed, and growth arrested cells. Under these conditions, thecells remained viable and retained their normal monolayer configurationand morphological appearance up to a concentration of 2 μg/mlhalofuginone. Na₂ [³⁵ S]O₄ (540-590 mCi/mmol) was added (40 μCi/ml) oneand five days after seeding and the cultures were incubated withoutmedium change. At various intervals after seeding, the subendothelialECM was exposed by dissolving (5 min., room temperature) the cell layerwith PBS containing 0.5% Triton X-100 and 20 mM NH₄ OH, followed by fourwashes in PBS [I Vlodavsky, et al., Cancer Res., Vol. 43, pp. 2704-2711(1983); I. Vlodavsky, et al., Proc. Natl. Acad. Sci. USA, Vol.84pp.2292-2296 (1987)]. To determine the total amount of sulfate labeledmaterial, the ECM was digested with trypsin (25 μg/ml, 24 h, 37° C.) andthe solubilized material counted in a β-counter.

FIG. 7 shows the almost complete inhibition of sulfate incorporation by1 μg/ml Halofuginone, while 50% inhibition was obtained in the presenceof 0.2 μg/ml of the drug.

EXAMPLE 6 Inhibition of Incorporation of Sulfate, Proline, Lysine andGlycine into ECM of Bovine Corneal Endothelial Cells

Corneal endothelial cells were seeded at a confluent density and grownas described in Example 5 above. The cells were cultured with or withoutHalofuginone in the presence of either Na₂ ³⁵ SO₄ (FIG. 8A), ³ H-proline(FIG. 8B), ¹⁴ C-lysine (FIG. 8C) or ¹⁴ C-glycine (FIG. 8D). Eight daysafter seeding, the cell layer was dissolved substantially as describedin Example 5 above. The underlying ECM was then either trypsinized todetermine the effect of Halofuginone on incorporation of labeledmaterial into total protein, substantially as described in Example 5above, or subjected to sequential digestions with collagenase andtrypsin to evaluate the effect of Halofuginone on bothcollagenase-digestible proteins (CDP) and non-collagenase digestibleproteins (NCDP).

As FIGS. 8A-8D show, Halofuginone inhibited the incorporation ofsulfate, proline, lysine and glycine into both CDP and NCDP, reflectinga profound inhibition of matrix deposition. The inhibitory effect ofHalofuginone on deposition of ECM components other than collagen is mostlikely due to the involvement of collagen in the assembly of otherconstituents into the supramolecular structure of the ECM.Alternatively, Halofuginone may affect the synthesis of ECM componentsother than collagen, possibly through a common transcription factor orcytokine such as TGFβ, which affects the synthesis and deposition ofseveral ECM components.

EXAMPLE 7 Inhibition of Sulfate and Glycine Incorporation into RatMesengial Cell ECM

Rat mesengial cells were grown to confluency, 24 hours after seeding.The cells were then cultured with or without Halofuginone in thepresence of either Na₂ ³⁵ SO₄ (FIGS. 9A and 9B) or ¹⁴ C-glycine (FIGS.9C and 9D). Eight days after seeding, the cell layer was dissolved toexpose the underlying ECM, washed and digested with collagenase todetermine the effect of Halofuginone on CDP proteins, as shown in FIGS.9A and 9C. The remaining material was digested with trypsin andsubjected to β-scintillation counting to determine the effect ofHalofuginone on NCDP proteins, as shown in FIGS. 9B and 9D.

About 30% inhibition of sulfate incorporation was seen for CDP proteins,while about 70% inhibition was seen for NCDP proteins in the presence of200 ng/ml Halofuginone. It should be noted that the inhibition of ECMdeposition by Halofuginone was not due to its anti-proliferativeactivity since the drug was added to highly confluent, non-dividingcells. Since inorganic sulfate is incorporated primarily into sulfatedglycosaminoglycans and not into collagen, it is conceivable that byinhibiting type I collagen synthesis, Halofuginone interferes with theassembly of other ECM macromolecules, such as heparin sulfateproteoglycans, which are known to specifically interact with collagen toform ECM.

About 80% inhibition of glycine incorporation was seen for both CDP andNCDP proteins in the presence of 50 ng/ml Halofuginone. The inhibitoryeffect of Halofuginone on deposition of collagenase-digestible ECMproteins was more pronounced with glycine than with sulfate labeledmatrix since unlike glycine, sulfate is incorporated primarily intoglucosaminoglycans which are not degraded by collagenase. A profoundinhibition of ECM deposition was supported by a microscopic examinationof the denuded culture dishes, revealing a thin or non-existant layer ofECM produced in the presence of Halofuginone.

EXAMPLE 8 Inhibitory Effect of Halofuginone on In VivoNeovascularization

Halofuginone was shown to inhibit angiogenesis in an in vivo model. Suchan inhibitory effect also demonstrates the ability of Halofuginone toinhibit cell proliferation which is enabled by the deposition of ECMcomponents. Results are given in FIGS. 10A and 10B, and in Table 3below.

A murine corneal angiogenesis model was used to evaluate the inhibitoryeffect of Halofuginone in vivo. The angiogenic factor bFGF was appliedinto a corneal pocket in a pellet made of a slow release polymer, asdescribed below, and Halofuginone (2 μg/mouse/day) was administered i.p.for 5 consecutive days.

The pellets were made of the slow-release polymer Hydron(polyhydroxyethylmethacrylate [polyHEMA], Interferon Sciences, N.J.)containing sucralfate alone, or sucralfate and bFGF. A suspension ofsterile saline 10 μg recombinant bFGF plus 10 mg of sucralfate wasprepared and speed vacuumed for 5 minutes. To this suspension 10 μl of12% Hydron in ethanol was added. The suspension was then deposited ontoan autoclave sterilized, 15×15 mm piece of nylon mesh (TETKO, 3-300/50,approximate pore size 0.4×0.4 mm) and embedded between the fibersresulting in a grid of 10×10 squares. Both sides of the mesh were thencovered with a thin layer of Hydron to hydrophobically stabilize thepellet during implantation. These layers were allowed to dry on asterile petri dish for 30 minutes. Subsequently, the fibers of the meshwere pulled apart under a microscope, and about 30-40 uniformly sizedpellets of 0.4×0.4×0.2 mm, containing approximately 80-100 ng bFGF perpellet, were collected. Such pellets can be stored frozen at -20° C. forseveral weeks without loss of bioactivity.

C57B1/6 mice of about 7-9 weeks of age were anesthetized withmethoxyflurane. The eyes were topically anesthetized with 0.5%proparacaine, and the globe was proptosed with a jeweler's forceps. Acentral, intrastromal linear keratotomy of approximately 0.6 mm lengthwas performed with a surgical blade and a lamellar micropocket wasdissected towards the temporal limbus. The temporal extent of the pocketwas within 0.7-1.0 mm of the limbus. A single pellet was then placed onthe corneal surface at the base of the pocket with jeweler's forceps,and using one arm of the forceps, the pellet was advanced toward thetemporal end of the pocket. Antibiotic ointment (erythromycin) wasapplied once to the operated eye, not only to prevent infection, butalso to decrease irritation of the irregular ocular surface. The eyeswere routinely examined by slit-lamp biomicroscopy on post-operativedays 3 through 5 following pellet implantation.

On post-operative day 5, mice were anesthetized with methoxyflurane, theeyes were proptosed, and the maximum vessel length (VL) of theneovascularization zone extending from the base of the limbal vascularplexus toward the pellet was measured with a linear reticule through theslit lamp. The contiguous circumferential zone of neovascularization(CH) was measured as clock hours with a 360 degree reticule. The eyeswere photographed on day 5 and the slides were used to determine thearea of neovascularization, in square mm.

The area of neovascularization was calculated according to the followingformula. ##EQU1## where A is area, CH is contiguous circumferential zoneof neovascularization and VL is maximal vessel length.

FIG. 10A demonstrates that neovascularization from the corneal limbus tothe pellet occurred in the eyes of control mice. The average area ofneovascularization in the control group was 2.66 mm² (n=8). FIG. 10Bshows that neovascularization was inhibited by Halofuginone, as theaverage area of neovascularization was 1.90 mm² (n=7), for an inhibitionof about 29%. Data for all mice are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Inhibition of Angiogenesis by Halofuginone                                      Sample   VL for   VL for    CH for CH for                                     No. Control Halofuginone Control Halofuginone                               ______________________________________                                        1      1.2      0.9         4      3                                            2 1.2 1 4 2.5                                                                 3 1.2 1.2 5 4                                                                 4 1.1 1 3 1                                                                   5 1.2 1 3 3.5                                                                 6 1 1.1 4 3                                                                   7 1.1 1.2 3 3                                                                 8 1 NA 4 NA                                                                 ______________________________________                                    

EXAMPLE 9 Effect of Halofuginone on Proliferation of HumanLeiomyosarcoma Tumor Cells

The effect of Halofuginone on proliferation of human leiomyosarcomatumor cells was investigated. Leiomyosarcoma tumors have abundantextracellular matrix and are also well vascularized [A. Ferenczy, etal., Cancer, No. 28, pp. 1004-1018 (1971)]. Their growth is thought tobe dependent on growth factors (i.e., bFGF, HB-EGF) produced by normaland malignant myometrial cells [A. Zhang, et al., Endocrinology, No.134, pp. 1089-1094 (1994); R. S. Mangrulker, et al, Biology ofReproduction, No. 53, pp. 636-646 (1995)] and locally embedded in thesurrounding ECM [I. Vlodavsky, et al., Basement Membranes: Cellular andMolecular Aspects, D. H. Rohrbach and R. Timpl, Eds., Academic Press,Inc., Orlando, Fla., U.S.A., pp. 327-343 (1993)].

Samples of human leiomyosarcoma tumors were obtained from womenundergoing surgical hysterectomy, as described [R. S. Mangrulker, etal., ibid]. Minced tissue was placed into 20 ml cold homogenizationbuffer: 1 M NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM benzamidine, 0.1%CHAPS, 0.01% Aprotinin (Sigma), 10 μg/ml leupeptin, 1 mM AEBSF [R. S.Mangrulker, et al., ibid.]. Samples were homogenized for 2 min andcentrifuged at 12000×g for 60 min at 4° C. The supernatants were diluted1:5 with 10 mM Tris (pH 7.4) to a final volume of 100 ml and filteredwith 0.45 μm nylon filters. Cells were plated and maintained in DMEMplus 10% calf serum. The cells remained viable over a number of passagesbut were used at passages 2 or 3.

For proliferation assays, cells were plated in 96-well plates at 10,000cells/well in 200 μl medium (DMEM with 4.5 g/L glucose, 10% calf serum,1% glutamine, 1% penicillin/streptomycin) and incubated 2 days untilconfluent. 24 h after seeding, increasing concentrations of Halofuginone(10-100 ng/ml) were added. The medium was changed to DMEM with 0.5% calfserum, 1 μM insulin and 5 μM transferrin. After 24 h, the samples (5-10μl) were added and after an additional 24 h, [³ H]thymidine (1 μCi/well)was added to each well. After 36-48 h incubation, the cells were fixedwith methanol, and the DNA was precipitated with 5% trichloroaceticacid. The cells were lysed with 150 μl/well of 0.3 N NaOH, transferredto scintillation vials, and counted on a β-counter. As demonstrated inFIG. 11A, 60-70% inhibition of [³ H]thymidine incorporation was obtainedat 2.5 ng/ml Halofuginone.

The effect of Halofuginone on proliferation of HB-EGF (Heparin-BindingEpidermal Growth Factor) and serum-stimulated leiomyosarcoma cells wasthen examined. The leiomyosarcoma tumor cells were growth-arrested by 48h incubation in medium containing 0.5% FCS. The cells were then exposed(24 h) to either 10% FCS or 10 ng/ml HB-EGF in the absence or presenceof 10 ng/ml Halofuginone. [³ H]thymidine was then added and DNAsynthesis measured 36 h later. A complete inhibition of cellproliferation induced by both serum or HB-EGF was observed in thepresence of Halofuginone, as shown in FIG. 11B.

EXAMPLE 10 Inhibition of Collagen Type I Gene Expression by Halofuginone

Myometrial and leiomyosarcoma cells were taken from the same patient andwere plated into 10 cm plates in DMEM supplemented with 10% FCS. Whenthe cells reached 80% confluence, the medium was replaced by serum freeDMEM plus 0.1% BSA for 48 hours, washed and exposed to increasingconcentrations of Halofuginone in the same medium for about 48 hours atabout 37° C. The cells were then harvested and subjected to RNAextraction and Northern blot analysis for collagen type I geneexpression. As demonstrated in FIG. 12, Halofuginone inhibited collagentype I gene expression (products at 5.4 and 4.8 kb) in a dose-dependentmanner. Furthermore, the effect on myometrial cells (lane M4, 50 ng/ml)as compared to leiomyosarcoma (lane L6, 200 ng/ml) cells.

EXAMPLE 11 Suitable Formulations for Administration of Halofuginone

Halofuginone can be administered to a subject in a number of ways, whichare well known in the art. Hereinafter, the term "subject" refers to thehuman or lower animal to whom Halofuginone was administered. Forexample, administration may be done topically (including ophtalmically,vaginally, rectally, intranasally), orally, or parenterally, for exampleby intravenous drip or intraperitoneal, subcutaneous, or intramuscularinjection.

Formulations for topical administration may include but are not limitedto lotions, ointments, gels, creams, suppositories, drops, liquids,sprays and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

Formulations for parenteral administration may include but are notlimited to sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Dosing is dependent on the severity of the symptoms and on theresponsiveness of the subject to Halofuginone. Persons of ordinary skillin the art can easily determine optimum dosages, dosing methodologiesand repetition rates.

EXAMPLE 12 Method of Treatment of Malignancies

As noted above, Halofuginone has been shown to be an effective inhibitorof tumor progression by inhibiting angiogenesis. The following exampleis an illustration only of a method of treating malignancies withHalofuginone, and is not intended to be limiting.

The method includes the step of administering Halofuginone, in apharmaceutically acceptable carrier as described in Example 12 above, toa subject to be treated. Halofuginone is administered according to aneffective dosing methodology, preferably until a predefined endpoint isreached, such as the absence of a particular tumor marker in a sampletaken from the subject.

Examples of tumors for which such a treatment would be effectiveinclude, but are not limited to, breast cancers such as infiltratingduct carcinoma of the breast, lung, cancers such as small cell lungcarcinoma, bone cancers, bladder cancers such as bladder carcinoma,rhabdomyosarcoma, angiosarcoma, adenocarcinoma of the colon, prostate orpancreas, squamous cell carcinoma of the cervix, ovarian cancer,malignant fibrous histiocytoma, skin cancers such as malignant melanoma,leiomyosarcoma, astrocytoma, glioma and heptocellular carcinoma.

EXAMPLE 13 Method of Manufacture of a Medicament Containing Halofuginone

The following is an example of a method of manufacturing Halofuginone.First, Halofuginone is synthesized in accordance with goodpharmaceutical manufacturing practice. Examples of methods ofsynthesizing Halofuginone, and related quinazolinone derivatives, aregiven in U.S. Pat. No. 3,338,909. Next, Halofuginone is placed in asuitable pharmaceutical carrier, as described in Example 11 above, againin accordance with good pharmaceutical manufacturing practice.

While the invention has been described with respect to a limited numberof embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

What is claimed is:
 1. A method for the treatment of a tumor sensitiveto the compounds below in a subject, comprising the step ofadministering a pharmaceutically effective amount of a compound having aformula: ##STR9## wherein: R₁ is a member of the group consisting ofhydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;R₂is a member of the group consisting of hydroxy, acetoxy and loweralkoxy, and R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl.
 2. A method according to claim 1, wherein saidcompound is Halofuginone.
 3. The method of claim 1, wherein the tumor isbreast cancer, lung cancer, bladder cancer, bone cancer, prostatecancer, pancreas cancer, cerix cancer, ovarian cancer, skin cancer, orleiomyosarcoma cancer.